Electric fields at the active site of an enzyme: direct comparison of experiment with theory

The electric fields produced in folded proteins influence nearly every aspect of protein function. We present a vibrational spectroscopy technique that measures changes in electric field at a specific site of a protein as shifts in frequency (Stark shifts) of a calibrated nitrile vibration. A nitrile-containing inhibitor is used to deliver a unique probe vibration to the active site of human aldose reductase, and the response of the nitrile stretch frequency is measured for a series of mutations in the enzyme active site. These shifts yield quantitative information on electric fields that can be directly compared with electrostatics calculations. We show that extensive molecular dynamics simulations and ensemble averaging are required to reproduce the observed changes in field.     

Acetylcholine receptor: covalent attachment of depolarizing groups at the active site

Following reduction of the acetylcholine receptor in the electroplax with dithiothreitol, the quaternary ammonium compounds bromoacetylcholine bromide and the p-nitrophenyl ester of (p-carboxyphenyl) trimethylammonium iodide react near the active site probably with a sulfhydryl group. The covalently attached quaternary ammonium moieties additionally interact with the active site noncovalently to activate the receptor and cause depolarization of the cell.     

Electrostatic and steric contributions to regulation at the active site of isocitrate dehydrogenase

The isocitrate dehydrogenase of Escherichia coli is regulated by covalent modification at the active site rather than, as expected, at an allosteric site. As a means of evaluating the mechanism of regulation, the kinetics of the substrate, 2R,3S-isocitrate, and a substrate analog, 2R-malate, were compared for the native, phosphorylated, and mutant enzymes. Phosphorylation decreases activity by more than a factor of 10(6) for the true substrate, but causes minor changes in the activity of the substrate analog. The kinetic results indicate that electrostatic repulsion and steric hindrance between the phosphoryl moiety and the gamma carboxyl group of 2R,3S-isocitrate are the major causes of the inactivation, with a lesser contribution from the loss of a hydrogen bond.     

Modification of the active site of alkaline phosphatase by site-directed mutagenesis

The catalytically essential amino acid in the active site of bacterial alkaline phosphatase (Ser-102) has been replaced with a cysteine by site-directed mutagenesis. The resulting thiol enzyme catalyzes the hydrolysis of a variety of phosphate monoesters. The rate-determining step of hydrolysis, however, is no longer the same for catalysis when the active protein nucleophile is changed from the hydroxyl of serine to the thiol of cysteine. Unlike the steady-state kinetics of native alkaline phosphatase, those of the mutant show sensitivity to the leaving group of the phosphate ester.     

Regulation of phospholipase C-gamma 1 by profilin and tyrosine phosphorylation.

Epidermal growth factor and platelet-derived growth factor can stimulate the production of the second messenger inositol trisphosphate in responsive cells, but the biochemical pathway for these signaling events has been uncertain because the reactions have not been reconstituted with purified molecules in vitro. A reconstitution is described that requires not only the growth factor, its receptor with tyrosine kinase activity, and the soluble phospholipase C-gamma 1, but also the small soluble actin-binding protein profilin. Profilin binds to the substrate phosphatidylinositol 4,5-bisphosphate and inhibits its hydrolysis by unphosphorylated phospholipase C-gamma 1. Phosphorylation of phospholipase C-gamma 1 by the epidermal growth factor receptor tyrosine kinase overcomes the inhibitory effect of profilin and results in an effective activation of phospholipase C-gamma 1.     

The manganese site of the photosynthetic water-splitting enzyme

As the originator of the oxygen in our atmosphere, the photosynthetic water-splitting enzyme of chloroplasts is vital for aerobic life on the earth. It has a manganese cluster at its active site, but it is poorly understood at the molecular level. Polarized synchrotron radiation was used to examine the x-ray absorption of manganese in oriented chloroplasts. The manganese site, in the "resting" (S1) state, is an asymmetric cluster, which probably contains four manganese atoms, with interatomic separations of 2.7 and 3.3 angstroms; the vector formed by the 3.3-angstrom manganese pair is oriented perpendicular to the membrane plane. Comparisons with model compounds suggest that the cluster contains bridging oxide or hydroxide ligands connecting the manganese atoms, perhaps with carboxylate bridges connecting the 3.3-angstrom manganese pair.     

Glucuronidase activation: enzyme action at an interface

Although the rate of hydrolysis by mammalian beta-glucuronidase appears to be inhibited by methylene chloride or carbon tetrachloride with the standard technique (phenolphthalein glucuronide as a substrate), the release of steroidal conjugates under conditions generally employed does not appear to be affected.     

Linking petrology and seismology at an active volcano

Many active volcanoes exhibit changes in seismicity, ground deformation, and gas emissions, which in some instances arise from magma movement in the crust before eruption. An enduring challenge in volcano monitoring is interpreting signs of unrest in terms of the causal subterranean magmatic processes. We examined over 300 zoned orthopyroxene crystals from the 1980-1986 eruption of Mount St. Helens that record pulsatory intrusions of new magma and volatiles into an existing larger reservoir before the eruption occurred. Diffusion chronometry applied to orthopyroxene crystal rims shows that episodes of magma intrusion correlate temporally with recorded seismicity, providing evidence that some seismic events are related to magma intrusion. These time scales are commensurate with monitoring signals at restless volcanoes, thus improving our ability to forecast volcanic eruptions by using petrology.     

Computational design of a biologically active enzyme

Rational design of enzymes is a stringent test of our understanding of protein chemistry and has numerous potential applications. Here, we present and experimentally validate the computational design of enzyme activity in proteins of known structure. We have predicted mutations that introduce triose phosphate isomerase activity into ribose-binding protein, a receptor that normally lacks enzyme activity. The resulting designs contain 18 to 22 mutations, exhibit 10(5)- to 10(6)-fold rate enhancements over the uncatalyzed reaction, and are biologically active, in that they support the growth of Escherichia coli under gluconeogenic conditions. The inherent generality of the design method suggests that many enzymes can be designed by this approach.     

Staphylococcal nuclease: size and specificity of the active site

The dissociation constants and standard free energies of complex formation determined with staphylococcal nuclease and a series of 5'-phosphoryloligothymidyl derivatives of increasing chain length suggest that maximum stability is reached with an oligonucleotide containing three nucleotide units. A proposed model of the active site that contains other knowledge of the specificity and the catalytic mechanism of this enzyme postulates the existence of three nonequivalent phosphate binding subsites and a closely related phosphodiester hydrolytic subsite.     

Dominance in physiological phenotypes and fitness at an enzyme locus

Aminopeptidase-I allozymes, which are products of the Lap locus in the marine mussel, Mytilus edulis, differ in their catalytic efficiencies. These biochemical differences result in genotype-specific rates of change in the free amino acid pool, that is, cell volume regulation, when mussels are subjected to changes in salinity. A high degree of dominance was found among genotypes for these biochemical and physiological phenotypes. Selection models that incorporate dominance adequately predict observed genotypic properties at the Lap locus among natural populations that exhibit clinical allele frequency. This suggests that a high degree of dominance for fitness must also occur at this locus in natural populations. These results provide additional evidence that the maintenance of an allele frequency cline is operating by natural selection at the Lap locus.     

Observation of covalent intermediates in an enzyme mechanism at atomic resolution

In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra-high resolution structures of wild-type and mutant d-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and (1)H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.     

Regulation of proteolysis at the neutrophil-substrate interface by secretory leukoprotease inhibitor

Human neutrophils can initiate the rapid degradation of extracellular matrix macromolecules by localizing the destructive process to sites of cell-substrate contact. Although plasma and its filtrates contain multiple proteinase inhibitors, these inhibitors did not prevent neutrophils from attacking either underlying fibronectin or elastin. However, subjacent substrates could be protected from neutrophils by recombinant secretory leukoprotease inhibitor, a structurally unique serine proteinase inhibitor whose natural counterpart is normally confined to human mucous secretions. The identification of this extravascular proteinase inhibitor as a potent regulator of subjacent proteolysis could lead to the development of a new class of anti-inflammatory therapeutics.     

A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase

An active site, cofactor-containing peptide has been obtained in high yield from bovine serum amine oxidase. Sequencing of this pentapeptide indicates: Leu-Asn-X-Asp-Tyr. Analysis of the peptide by mass spectrometry, ultraviolet-visible spectroscopy, and proton nuclear magnetic resonance leads to the identification of X as 6-hydroxydopa. This result indicates that, contrary to previous proposals, pyrroloquinoline quinone is not the active site cofactor in mammalian copper amine oxidases. Although 6-hydroxydopa has been implicated in neurotoxicity, the data presented suggest that this compound has a functional role at an enzyme active site.     

污泥处理渣对电子废弃物拆解污染土壤的改良作用

为探究两种污泥处理渣对电子废弃物拆解污染土壤的改良作用,通过盆栽试验解析添加热解渣和好氧发酵渣对土壤养分、酶活性、重金属浸出与植物（黑麦草和香根草）生长、吸收重金属的影响,并通过模糊隶属函数评价土壤改良效果。结果表明：施用两种污泥处理渣（施用量均为5%）向土壤引入了类胡敏酸物质;显著提高土壤电导率（220%~238%）、铵态氮（16%~66%）、阳离子交换量（37%~84%）、有效磷（34%~42%）、速效钾（44%~97%）、有机质（17%~47%）含量和荧光素双醋酸酯（FDA）酶活性（67%~119%）;显著提高植物鲜质量（32%~36%）、株高（18%~22%）及叶绿素（12%~16%）、磷（33%~48%）和钾（12%~21%）含量;显著降低植物Cu（17%~33%）、Zn（5%~9%）、Pb（32%）、Cd（35%）和丙二醛（18%~22%）含量;显著降低土壤Cu（10%~19%）、Zn（18%~20%）、Pb（10%~16%）和Cd（13%~15%）的浸出量。土壤改良效果的排序为好氧发酵渣>热解渣,香根草>黑麦草。研究表明,施用两种污泥处理渣均促进了植物生长和土壤改良,其中好氧发酵渣和香根草在电子废弃物拆解污染土壤生态修复领域具有更佳的应用潜力。